Hydrogen peroxide (HO) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with HO activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of HO to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of HO to increase paracellular permeability. HO treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. HO treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, HO increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to HO was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. HO treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that HO increases MDCK cell paracellular permeability through activation of ERK 1/2. This HO action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

Renal epithelial tight junction permeability to large molecules (leak pathway) is increased by hydrogen peroxide (H2O2). Previous studies have suggested a role for the actin cytoskeleton in regulation of epithelial paracellular permeability but its possible involvement in H2O2‐induced regulation of renal epithelial paracellular permeability is unknown. To assess the role of the actin cytoskeleton, we examined the effect of two agents that manipulate differentially the actin cytoskeleton, blebbistatin (inhibits myosin II ATPase) and jasplakinolide (enhances F‐actin nucleation), on basal and H2O2‐induced renal epithelial leak pathway permeability, measured as calcein flux rate. The effects of these compounds was examined in wild type MDCK cells and in MDCK cells in which either occludin protein or ZO‐1 protein has been knocked down to determine if either of these tight junction proteins might be important in mediating actin network actions. Under basal conditions, treatment with 40 mM blebbistatin produced little or no change in calcein flux rate in wild type MDCK cells. H2O2 increased calcein flux rate, as expected. The combination of H2O2 and blebbistatin produced a synergistic increase in calcein flux rate. Jasplakinolide (200 nM) produced a marked increase in calcein flux rate. H2O2 and jasplakinolide did not increase calcein flux rate beyond the rate produced by jasplakinolide alone. Knockdown of occludin protein in MDCK cells did not affect basal calcein flux rate but enhanced the H2O2‐induced increase. In the absence of H‐2O2, blebbistatin produced little or no change in calcein flux rate. H2O2 and blebbistatin did not increase calcein flux rate further than that produced by H2O2 alone. Jasplakinolide increased the calcein flux rate but attenuated the increase produced by H2O2 when cells were incubated with both compounds. Knockdown of ZO‐1 protein increased basal calcein flux rate approximately two‐fold. Treatment with H2O2 did not further increase flux rate. Treatment of ZO‐1 knockdown cells with blebbistatin increased the calcein flux rate, in contrast to its lack of effect on wild type MDCK cells. Treatment with the combination of H2O2 and blebbistatin did not increase flux rate beyond that produced by blebbistatin alone. Jasplakinolide alone increased calcein flux rate in ZO‐1‐knockdown cells. The combination of H2O2 and jasplakinolide did not increase flux rate beyond that produced by jasplakinolide alone. These results suggest a complex role of the actin cytoskeleton in regulation of basal and H2O2‐stimulated leak pathway permeability in renal epithelial cells. First, they suggest that an intact F‐actin network is necessary to maintain low basal leak pathway permeability. Second, they suggest the tight junction is under inward actomyosin‐mediated tension, via the ZO‐1 protein, which contributes to maintenance of low leak pathway permeability, particularly in stress conditions. Third, they suggest that H2O2 treatment produces a state in MDCK cells that is functionally similar to knockdown of ZO‐1 protein. Fourth, they suggest that the effect of blebbistatin is mediated via occludin protein.Support or Funding InformationNIDDK and institutional fundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Based on a previously developed model, which included maternal age, BMI and cervical exam on admission, gestational age, race/ ethnicity, any prior preterm or term VD, type of labor and PROM, we calculated the predicted probability of VD for each woman, and estimated the prevalence of maternal and neonatal morbidity according to this predicted probability. RESULTS: The 45,997 women in this analysis met eligibility criteria and had all variables needed to calculate probability of VD. More than 75% of the study population had a predicted probability of VD >90%. Maternal and neonatal morbidity became less frequent as the predicted probability of VD increased. For example, when the probability of VD was > 95%, less than 4% of women experienced severe morbidity compared to approximately 20% when the probability was < 80% (Table). CONCLUSION: A model to predict VD in women at term with singleton gestations is also associated with maternal and neonatal morbidity. If validated, this VD calculator may be clinically useful in stratifying patients according to their probability of VD and perinatal morbidity, which may be useful to direct staffing and to adjust for case-mix when comparing rates of morbidity between various systems.